Vitamin A and some of its analogs (retinoids) prevent squamous differentiation of normal nonkeratinizing epithelial cells in vivo and in vitro and suppress oral premalignant lesions (leukoplakia) in man and carcinogenesis in the upper aerodigestive tract in experimental animals. The mechanism(s) by which retinoids exert these effects is not known. Nuclear retinoic acid receptors (RARs) function as ligand-activated trans-acting transcription modulating factors and mediate the effects of retinoids on the expression of certain genes. The hypotheses underlying this project are: (A) Retinoids' ability to suppress malignant cells, and (B) These effects result from changes in gene expression mediated via nuclear retinoid receptors. The objectives of this project are: To determine the relevance of the RARs for mediation of the effects of retinoids on the growth and differentiation of human head and neck squamous cell carcinoma (HNSCC) cells in vitro. Three HNSCC cell lines before and during treatment with 13-cis retinoic acid (13cRA) in vitro and biopsies of human oral mucosa and leukoplakia lesions taken from patients before and during treatment with 13cRA will be used to: (a) Determine the presence and level of RAR-alpha, RAR-beta, RAR-gamma, and RXR-alpha in HNSCC cells at the mRNA level (by Northern blotting) and at the protein level (by immunoblotting and radioactive retinoid binding); (b) Determine whether the expression of any of the RARs is modulated in HNSCC cells by retinoid treatment; (c) Determine whether the level of any of the RARs before and after retinoid treatment is related to the responsiveness of the HNSCC cells to the effects of 13cRA on squamous cell differentiation markers transglutaminase, involucrin, and K1 keratin at the mRNA level and at the protein level; (d) Determine whether blocking the expression of one or more of the RARs by antisense oligonucleotides or mRNA will inhibit any of the effects of 13cRA on the HNSCC cells; (e) Determine the presence and level of RARs and the expression of transglutaminase, involucrin, and K1 keratin in "normal" mucosa cells and in biopsies from oral leukoplakia lesions before and after treatment with 13cRA in vivo by in situ hybridization with nucleotide probes for RARs' and differentiation markers' mRNAs, and immunohistochemical analysis with antibodies to each receptor and differentiation marker. These studies are expected to provide important information on the involvement of RARs in the mechanism of action of retinoids on HNSCC in vitro and possibly on premalignant oral epithelial cells in vivo.